Prader-Willi syndrome (PWS) is a multisystem disorder caused by loss of function of a cluster of ~12 paternally-expressed, imprinted genes. Cardinal features of PWS include neonatal failure to thrive, abnormal body composition, short stature with growth hormone deficiency, and childhood-onset hyperphagia and obesity, among other endocrine and behavioral abnormalities. Although mouse models of PWS recapitulate some of the clinical components of the disorder, they do not develop early- onset hyperphagia or the severe obesity of the human disease. Therefore, alternative animal models are needed to study the biomedical basis and therapeutic approaches for the pleiotropic clinical features of the disorder, especially the eating disorder and obesity. We propose that miniature pigs (minipigs) are ideal to generate a model for PWS. Technologies exist in the minipig to produce genetic models of disease. Most importantly, they have a more similar body size, physiology, anatomy, and genome to human than does the mouse. As a result, we hypothesize that minipigs with PWS will be more susceptible to development of obesity than the corresponding mouse models. We have recently generated a clone map and the DNA sequence for the pig PWS-orthologous region, defining the major regulatory elements that control imprinting and all the imprinted genes in the PWS- domain. Thus, the genomic tools are now in place to produce a porcine model of PWS. Specific Aim 1 of this application is to use a TALEN (Transcription Activator-Like Effector Nuclease) approach in male and female fetal pig cells to target a deletion of the imprinting control (IC) region on the maternal allele. Specific Aim 2 is to use somatic cell nuclear transfer to generate animals. Targeting in male cells and paternal transmission will quickly provide a PWS-model, allowing confirmation of the utility of a pig model to study hyperphagia and obesity; however, these pigs may be infertile as is the case in human PWS. By targeting the silent maternal allele in female cells, we will be able to maintain the mutation in a normal, female carrier pig line that only after paternal transmission of the mutation will produce litters of PWS and wildtype control piglets for clinical, pathophysiological, and molecular studies. A porcine model of PWS will lead to an understanding of hyperphagia and obesity in PWS and provide a means for therapeutic testing. Additionally, this work will set the stage to determine how biological pathways in PWS relate to genetic, dietary, and behavioral causes of human obesity and known components of body weight homeostasis.